由美国国立卫生研究院授予P20 RR16440的子项目和桥从西弗吉尼亚大学的研究和研究生教育厅授予南杂草支持。 L.洛佩斯 - 斯金纳在项目开发的早期阶段的技术援助表示感谢。作者也感谢提供技术援助和OSC19细胞J. Myers和M.尤尼斯（头颈外科部，MD安德森癌症中心，休斯敦，德克萨斯）; P.特纳和K. Secrest（西弗吉尼亚大学部病理组织银行）为组织学处理和程序，R. Wysolmerski（西弗吉尼亚大学神经生物学和解剖学系）pLL7.0慢病毒载体的LifeAct mCherry建设和J.熊（北卡罗莱纳大学）。西弗吉尼亚大学显微成像设施的使用（由美国国立卫生研究院授予P20 RR16440和玛丽Babb兰多夫癌症中心的支持）和非线性光学显微镜实验室（NLOM;由西弗吉尼亚大学神经科学中心之间的联合协作和支持西弗吉尼亚大学物理学部/西弗吉尼亚州的纳米技术倡议）也表示感谢。由美国国立卫生研究院授予P30 RR031155 NLOM是支持神经科学中心的一部分。

1。细胞株中，载体的构建及慢病毒生产<ol><li>在培养人的头部和颈部肿瘤细胞系（OSC19或UMSCC1）组成的完整的媒体的DMEM（Cellgro猫＃50 - 003 - PB）与10％小牛血清胎牛血清（Hyclone公司猫＃SH30070.03），1％青霉素补充/链霉素（Cellgro猫＃30 - 002 - CI），1％非必需氨基酸（Cellgro猫＃25 - 025 - CI）。 </li><li>要转移到的LifeAct mCherry编码序列的pLL7.0慢病毒载体，在父mCherry的cDNA Sbf1识别位点改变​​引入定点突变（Stratagene公司猫＃200518-5）的识别序列的3个沉默突变。修改LifeAct mCherry序列PCR扩增与侧翼EcoR1/Sbf1网站和亚克隆到pLL7.0产生的pLL7.0 LifeAct mCherry构造。 </li></ol> 2。 pLL7.0 - LifeAct mCherry病毒生产<ol><li>病毒生产慢病毒表达系统手册（系统生物科学版2-051018）。 </li><li>包装细胞系293T/17细胞（ATCC猫＃CRL - 11268）增长到40％，在头颈部鳞癌线使用相同的完整的媒体合流。 </li><li>细胞转染的pLL7.0 LifeAct - mCherry，psPAX2，并在3:2:1的比例pVSV - G的载体，分别使用CalPhos（Clontech公司猫＃631312）。 </li><li> 24小时后，从转染的初步媒体与新媒体取代。 </li><li>媒体当时收集和补充为72小时，每12小时，并保存在4 ° C </li></ol> 3。头颈细胞系的生产与稳定的LifeAct mCherry表达<ol><li>收集媒体是在2000年的10分钟的转速旋转4 ° C。 </li><li>一毫升含有病毒的澄清媒体直接添加OSC19或UMSCC1细胞12小时。细胞，然后冲洗干净，并为另外12个小时的时间内增加一个额外的病毒之一毫升。 </li><li>细胞用含有两个星期200mg/ml嘌呤霉素选择抗性菌落媒体。 </li><li>尚存的克隆筛选LifeAct mCherry表达直观地通过荧光显微镜观察。个别阳性菌落，用无菌3毫米克隆光盘（费舍尔猫＃0790710A）typsinized。 </li><li>阳性细胞维持，直到冻结或原位注射含200mg/ml嘌呤媒体。 </li></ol> 4。原位肿瘤异种移植形成<ol><li>按照西弗吉尼亚大学动物护理和使用委员会批准了一项协议（09-0821），所有动物的程序进行。 </li><li> LifeAct - mCherry表达的肿瘤细胞，胰酶消化，离心，2.5 × 10 4细胞重悬于50μL完整的媒体。 </li><li>肿瘤细胞装入一个连接到一个27毫升的注射器半针头。 </li><li>女裸鼠Fox1 NU / nu小鼠8周龄（哈伦实验室）80mg/kg氯胺酮和10mg/kg的甲苯噻嗪联合麻醉。加热垫，麻醉小鼠均保持在37-40 ° C之间。 </li><li>用无菌镊子，舌尖轻轻抓住，小心拉出口腔。 </li><li>细胞注入每个舌的一侧慢慢在舌中心创建一个球茎质量，避免舌动脉。 </li><li>小鼠注射2.1mg/kg育亨宾和返回他们在那里为2-3小时监控从麻醉中恢复期间的加热垫。 </li><li>一旦恢复，小鼠放入含有软转基因面团饮食（S3472＃BioServe公司猫）无菌笼。 </li><li>小鼠称重，每2-3天，可视化和监测肿瘤发病。 </li></ol> 5。 EX -活体成像的小鼠舌头的制备<ol><li>窝藏在不同时间点（通常注射后2-4周）肿瘤的小鼠吸入二氧化碳安乐死。 </li><li>提取舌头，用1X PBS漂洗，并连接到一个传统的石蜡组织包埋盒式侧（StatLab猫＃H104）从本地爱好店和一个大小为8缝衣针使用的单丝缝纫线。 </li><li>一旦被固定的舌头，整个磁带大会被安置在30毫米组织培养皿中，并沉浸在1X PBS。 </li><li>加工舌头立即用于双光子激发显微镜。 </li></ol> 6。双光子显微镜成像舌肿瘤<ol><li>舌录音带被淹没在一个60毫米含有1X PBS菜定制设计一个可伸缩的悬臂下一个堂堂正正的显微镜（可移动的显微镜（MOM），萨特仪器）目标定位（商会班车，锡斯基尤文书）的持有人担保。 </li><li>一个40X/0.8NA水浸渍物镜直接放置或以上可见慕尼黑或病变。舌头与钛蓝宝石激光器（MIRA，连贯）优化mCherry信号强度为60兆瓦，输入波长755纳米的双光子显微镜成像。 </li><li>串行1毫米激光扫描图像采集在1微米的深度超过15和100微米之间（取决于肿瘤体积）共组织深入增量。被抓获的图像使用ScanImage基于MATLAB平台，是由卡雷尔斯沃博达实验室（Janelia农场，HHMI）的开发，开放源码的程序。 ScanImage产生的光栅扫描模式的双通道输出控制X / Y振镜扫描镜，并在同一时间捕捉一个最大的的四通道信号输入，同时通过数据采集板的光电倍增管（光电倍增管）（PCI - 6610S ，美国国家仪器公司）。光电倍增管的信号放大，低噪声电流前置放大器（SR570，斯坦福研究系统），在喂养的NI - DAQ板，显示在监视器屏幕上。 ScanImage收集控制目标的Z轴的Z - Stack的图像，并收集在一个单一的或循环模式的时间推移图像。图像保存在一个单一的TIFF文件，16位深度。 </li></ol> 7。使用阿米拉软件进行图像分析<ol><li>阿米拉成像对肿瘤量化：在Aimra软件中，打开包含Z - Stack的图像的TIFF文件。 </li><li>突出显示的文件名称，选择和应用Voltex功能生成三维渲染。一个大主与几个较小的，分离侵入组集体入侵细胞（IG）肿瘤形象呈现的图像中通常明显（见图6A）。 </li><li>要选择主肿块，选择“打开数据”，然后在“标签”，那么“标签字段”。 </li><li>阈值的原发肿瘤，选择所有的Z - Stack的图像，并通过在z平面的图像，滚动，以确保在三个方面列入只有小学肿块。这通常是该领域最大的尺寸的图像，并常常出现分割的成像是通过z平面走过。 </li><li>使用魔术棒/阈值的功能，以正确的背景荧光，消除它从没有丢弃任何的肿瘤信号形象。突出显示“内部文件”标签，并单击⊕按钮。选择“所有切片”复选框。这将产生一个大约每Z - Stack的增量的阈值在原发肿瘤区域的彩色边框。 </li><li>要选择IGS，选择“新建”功能。选择一个IG和确保它不是通过滚动z平面的原发肿瘤相关。 </li><li>选择“所有切片”，并单击⊕按钮。重命名文件（例如：IG 1） </li><li> 7.4重复阈值的程序，并在7.5突出确定IG一步。选择一个轮廓的颜色比，用来表示主肿块颜色不同。 </li><li>重复整个图像的每个额外的中空玻璃的必要，通过Z -平面扫描，以确保所有IGS表示。使用不同的颜色为每个确定的中空玻璃艾滋病在未来的形象识别。 </li><li>一旦原发肿瘤和所有IGS的选择，从下拉菜单中选择“分割”，然后选择“资料统计”。这提供了体积测量以及X，Y和X肿瘤原发肿瘤的核心坐标，从中央的肿瘤点，以计算距离的IGS。 </li><li>随着卷计算，确定每个IG的距离从原发肿瘤的核心。选择“分割”，“资料统计”。这一步计算成像素的所有测量。将像素转换基于显微镜物镜的校准和放大倍率任何额外增加（即变焦功能）微米。显微镜，并在这些实验中使用的设置，使用40X的目标是没有变焦，给予1个像素= 0.298微米的校准。数据导入到Excel电子表格，这给在三个方面的主要肿瘤（X1，Y1，Z1），并为每个IG（X2，Y2，Z2为第一IG前）的参数。 </li><li>从原发肿瘤中心为每个中空微米的入侵距离计算公式√（（X2 - X1的）2 +（Y2 - Y1）2 +（Z2 - Z1）2） 。 </li><li>肿瘤浸润指数（T）计算公式T I = N T x垂直T X D T N T = IGS在图像的总数，V T =所有IGS的总体积 ， D T =总行驶距离从原发肿瘤中心的所有侵入组。 </li></ol> 8。三与尼康的NIS - Elements软件的三维效果图<ol><li>三尼康NIS - Elements软件导入原来的16位单色TIFF整个肿瘤图像文件，可以产生更大的地形细节的三维渲染包（尼康，梅尔维尔，纽约州）。选择“文件”，然后“ND”，然后“创建文件序列的Nd文件”。选择TIFF Z系列的形象栈，并指定相应的步骤大小。 </li><li>校准在XY平面ND文件。指定的一个像素使用手动校准的大小。 </li><li>选择“卷视图”。使用“总部”的功能，更高的质量来计算z平面的其他片。在“3D渲染设置”，使用“高级渲染”与“超详细”和“全分辨率”的质量。选择“Alpha混合”，以突出肿瘤表面。 </li><li>优化用于演示的三维图像。在肿瘤和相关的IGS和作物需要放大。在XYZ平面旋转的图像和调整的LUT。 </li><li>捕捉演示的图像。选择“编辑创建视图快照（8位RGB）”。相应的名称和保存文件。 </li></ol> 9。代表性的成果<img src="/files/ftp_upload/2941/2941fig1.jpg" alt="图1" /> 图1。整体示意图，说明原位舌肿瘤的生产和原位双光子成像的关键步骤。 <img src="/files/ftp_upload/2941/2941fig2.jpg" alt="图2" /> 图2。LifeAct - mCherry OSC19细胞注射到老鼠的母语表达。 <img src="/files/ftp_upload/2941/2941fig3.jpg" alt="图3" /> 图3切除肿瘤含小鼠舌准备两个光子成像。 <img src="/files/ftp_upload/2941/2941fig4.jpg" alt="图4" /> 图4。定位在位置上的双光子显微镜成像肿瘤含舌。 <img src="/files/ftp_upload/2941/2941fig5.jpg" alt="图5" /> 图5代表屏幕上拍摄的，从ScanImage展示最初的双光子图像采集原始数据。 <img src="/files/ftp_upload/2941/2941fig6.jpg" alt="图6" /> 图6：在原位OSC19肿瘤，肿瘤侵袭的图像分析和定量。代表屏幕上拍摄的图像阿米拉Voltex渲染（A）和一个单一的阈值的Z -第（二）与主要的原发肿瘤紫色，侵入在蓝组1概述（IG - 1）概述，概述了红色和IG IG - 2 -3概述了绿色的识别过程中的一个例子。箭头表示IGS在（A）和（B）卷 C图与八个单独用来计算肿瘤侵袭指数（TI）IGS入侵距离。 <img src="/files/ftp_upload/2941/2941fig7.jpg" alt="图7" /> 图7。肿瘤的代表图像，并从本议定书入侵肿瘤组与常规免疫组化方法的图像相比 。A.鼠标整个舌头，窝藏UMSCC1原位肿瘤的石蜡切片。免疫组织化学染色是使用常规程序进行对特定的头颈部鳞癌细胞标记EMMPRIN（Zymed公司;猫＃34-5600）的主要抗体，1:1000稀释和使用OmniMap民建联反RB检测试剂盒（Ventana公司的猫＃760的可视化-149），其次是铁苏木素染色。包括舌头总面积的图像单独收集奥林巴斯ZX70 Provis光电MicroFire CCD相机显微镜放大4X和重建使用StereoINvestigator成像包（医疗保险基金生命科学）。肿瘤是显而易见的，在舌尖。扩大区域入侵前和个人的肿瘤细胞群（箭头）（B）C.尼康NIS元素渲染的具有代表性的OSC19肿瘤。增强的轮廓细节和清晰的可视化的IGS（箭头）是显而易见的。所有图像酒吧= 100微米。

<ol>
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原位小鼠模型为研究的头部和颈部癌症1,2许多方面已证明是有益的。我们结合双光子显微成像mCherry标记的细胞，作为一个系统来研究的头部和颈部肿瘤细胞浸润的早期事件以及建立原位系统的SCOT 3 。在此过程中，我们注意到，细胞可以从肿瘤注射部位泄漏，尤其是在小鼠的6个星期或以下由于没有足够的舌头大小，。我们用年纪较大的老鼠，以避免这个问题。与年纪较大的老鼠的舌头的大小，也有助于避免了舌动脉和舌过度出血破裂。肿瘤采取的是大大提高时，舌头在初始注射部位急剧膨胀。这种肿胀消退注入流体一两个小时内被吸收。肿瘤的生长出现明显的一两个星期注射后表示注入细胞数作为一个小舌头表面上的白色凹凸。我们还注意到，在这里介绍我们的研究中使用的40X目标，我们可以不区分个别的肿瘤细胞，但也确定为侵入性的细胞团，总结头颈部鳞癌侵袭的典型模式IGS。 迄今为止，这种模式已被广泛用于特定分子的作用，以及一些抗肿瘤的药物SCOT增长入侵，疗效监测颈部淋巴结转移，采用免疫组化或发光的方法4-7测量与测试。这个系统清单接近舌面上（图7）在肿瘤形成，使双光子显微镜的应用程序在侵入性的多细胞集群形象整个肿瘤原位。该程序也可以利用可视化与肿瘤细胞的决议的入侵。双光子显微镜先前已经利用原位8,9和异种移植8,10模型的头部和颈部癌症的研究试验性治疗。然而，这些报告和我们的协议之间的主要区别有两个。首先，这些研究使用外的标签，以目标/确定头部和颈部肿瘤细胞，可能限制与检测肿瘤细胞获得充足的流通。二，入侵肿瘤细胞接近到主站点，可能重演早期转移性活动是不是作为一个实验参数进行评估。我们的协议提供了在任何时候直接量化鼠标舌头肿瘤的进展过程中肿瘤细胞的侵袭能力。虽然这里介绍的方法使用解剖舌头描述的过程，我们目前在适应这种方法结合使用生物发光的活体小鼠的图像肿瘤的侵袭，同时监测早期的入侵，当地的淋巴结转移和远处转移的过程相同的动物。改建为活体成像的协议需要设计一个适当的定位阶段和维护过程中成像小鼠，以及作为一个实用的系统，以适当的灌溉口腔麻醉小鼠，在成像过程。一旦优化，这些适应化修改，将提供的学习能力，潜在的亲创分子和抗侵袭，局部浸润和更遥远的参与更长的时间内，在动物身上转移化合物的测试的作用。

multi-photon imaging of tumor cell invasion in an orthotopic mouse model of oral squamous cell carcinoma

局部区域的头部和颈部癌症的侵袭与转移风险，并提出了一个艰巨的挑战，在设计和实施病人的管理策略。口腔癌的原位小鼠模型已发展促进因素的研究评估抗肿瘤疗法的影响作为模型系统的入侵和服务。在这些系统中，可视化口腔组织内扩散的肿瘤细胞通常被无论是传统的组织学或在体内的生物发光方法进行。这些技术的主要缺点是固有的无法准确可视化和量化早期肿瘤细胞的侵袭，在三维空间中的主站点所产生的。在这里，我们描述了一个协议，结合舌鳞状细胞癌（SCOT）建立了双光子成像模型允许多矢量舌肿瘤扩散的可视化。 OSC - 19头颈部肿瘤细胞株的稳定设计，来表达的F -肌动蛋白结合肽LifeAct融合mCherry荧光蛋白（LifeAct mCherry）。可靠Fox1 NU / NU与这些细胞注入小鼠的肿瘤形成，使舌头前体内应用双光子显微镜观察。这种技术可以让肿块原位可视化和本地切除舌头侵略无区域的肿瘤微环境的破坏细胞。此外，这个系统可以让肿瘤细胞的侵袭入侵细胞从原发肿瘤部位移动的距离计算的量化。总体来说，这一过程提供了一个分析SCOT入侵和定制，以防止局部浸润和远处转移扩散的治疗因素，有助于增强型系统。这种方法也有可能最终要与其他成像方式在体内设置相结合的。

Watch this Scientific Journal Video about Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma at JoVE.com

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

产生的口腔癌和肿瘤浸润的定量监测的小鼠模型，通过多光子显微镜标记细胞内的舌头所涉及的技术的一个全面的概述。该系统可以作为一个有用的平台和反侵入性化合物的分子评估药物疗效。

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

multi-photon-imaging-tumor-cell-invasion-an-orthotopic-mouse-model

Research

JoVE Journal

Medicine

18.9K 次观看.  Mary Babb Randolph Cancer Center, West Virginia University. 产生的口腔癌和肿瘤浸润的定量监测的小鼠模型，通过多光子显微镜标记细胞内的舌头所涉及的技术的一个全面的概述。该系统可以作为一个有用的平台和反侵入性化合物的分子评估药物疗效。

视频: Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

一个小鼠膀胱癌原位模型

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

科研

医学

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

在原位小鼠脑肿瘤模型结合放疗

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

3维建模和成像集体细胞的侵袭

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

原位膀胱肿瘤模型和评价治疗膀胱萨纳

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

甲状腺未分化癌的原位小鼠模型

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

角膜新生血管在小鼠碱烧伤模型

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current &#39;gold standard&#39; method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The presence of high-risk HPV in head and neck tumor tissue is associated with favorable outcomes. The recently developed RNA in situ hybridization technique called RNAscope allows direct visualization of HPV E6/E7 mRNA in FFPE tissue sections.

RNAscope的<em&gt;原位</em&gt;检测转录活性的人类乳头状瘤病毒在头颈部鳞状细胞癌

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

原位胰腺癌小鼠模型的动态对比增强磁共振成像

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

“以病人为像”肝癌原位同源小鼠模型的癌转移

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...